CN117304267B - 一种扇贝鲜味肽及其制备方法与应用 - Google Patents
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Abstract
本发明属于食品生物技术领域,公开了一种扇贝鲜味肽及其制备方法与应用。本发明的扇贝鲜味肽获取自海湾扇贝,其包括氨基酸序列如SEQ.ID.NO.1~4所示的肽中的至少一种。本发明的扇贝鲜味肽通过两步酶解获得。本发明获得的鲜味肽有较低的鲜味阈值以及较强的增鲜提鲜作用。本发明的鲜味肽可用于调味品制作,改善食品风味,具有广阔的市场前景。
Description
技术领域
本发明属于食品生物技术领域,具体涉及一种扇贝鲜味肽及其制备方法与应用。
背景技术
含有鲜味的日常食物很多,如鱼、肉、贝类和酱油等。这些食品鲜味物质主要包括有机酸、鲜味肽、核苷酸和某些氨基酸。特别重要的是,鲜味肽营养丰富,无抗原性,易于吸收,可以增强食物的醇厚口感和香气。鲜味肽作为一种理想的天然、安全调味物质,近年来成为研究热点。
目前报道的鲜味肽大多集中在3000 Da以下,多数由蛋白质水解或微生物发酵法获得,也有少量通过提取或化学合成获得。其中,酶水解法是制备鲜味肽最常用的方法。酶解获得鲜味肽需要严格控制水解时间、温度、酶使用量等反应条件,否则难以获得稳定的产物,甚至产生苦味等不良风味;且酶解获得的产物是复杂的混合物。
鲜味肽可以通过与鲜味受体相互作用,激活味觉系统级联信号传递使得大脑感知到鲜味。T1R1/T1R3是G蛋白偶联受体家族成员,作为异源二聚体,被认为是最好的鲜味受体。近年来,同源性建模和分子对接被广泛用于揭示鲜味肽与其受体T1R1/T1R3之间的结合位点,为鲜味肽的筛选提供了有效途径。
海湾扇贝(Argopecten irradians)是扇贝科海湾扇贝属贝类。海湾扇贝对盐度和温度的适应范围广,滋味鲜美,是鲜味肽的优良来源。在海湾扇贝中提取鲜味肽,探究扇贝鲜味肽与鲜味受体T1R1/T1R3间的相互作用,阐明其具体结构,揭示其呈鲜机理,能够为海湾扇贝的高值化利用提供新途径。
发明内容
本发明提供一种扇贝鲜味肽及其制备方法与应用。本发明鲜味肽从海湾扇贝中分离提取得到,有较低的鲜味阈值以及较强的增鲜提鲜作用。
具体技术方案如下:
本发明的目的之一是提供一种扇贝鲜味肽,其包括氨基酸序列如SEQ.ID.NO.1~4所示的四种肽中的至少一种。
其中,SEQ.ID.NO.1为APDFGNR,该鲜味肽与鲜味受体T1R1/T1R3对接能量为-100.45 kcal/mol。
其中,SEQ.ID.NO.2为RGFGGAR,该鲜味肽与鲜味受体T1R1/T1R3对接能量为-102.943 kcal/mol。
其中,SEQ.ID.NO.3为RPPVVR,该鲜味肽与鲜味受体T1R1/T1R3对接能量为-81.3746 kcal/mol。
其中,SEQ.ID.NO.4为SWLDGK,该鲜味肽与鲜味受体T1R1/T1R3对接能量为-90.3756 kcal/mol。
具体地,所述的扇贝为海湾扇贝,上述鲜味肽分离提取自海湾扇贝的贝肉。
本发明的目的之二是提供上述鲜味肽的制备方法,其包括如下步骤:
S1. 获取扇贝肽;
S2. 对扇贝肽进行序列鉴定;
S3. 筛选可与鲜味受体T1R1/T1R3有效结合的扇贝肽。
进一步,步骤S1中:通过对扇贝的贝肉进行酶解获得扇贝肽。
具体地,步骤S1中:依次使用内切酶和外切酶对扇贝的贝肉进行酶解。
其中,所述的内切酶优选为碱性蛋白酶。
其中,所述的外切酶为风味蛋白酶。
更具体的,酶解的工作条件优选如下:向待处理原料中加入碱性蛋白酶,调节pH 8~10,30~40 ℃酶解5~7 h,灭酶;冷却后,加入风味蛋白酶,调节pH 6~7,50~60 ℃酶解5~8h,灭酶。
其中,碱性蛋白酶的添加量优选为以贝肉计1500~2000 U/g。
其中,风味蛋白酶的添加量优选为以贝肉计500~1000 U/g。
具体地,步骤S1中:在对贝肉进行酶解前,对其进行预处理。所述的预处理包括:向贝肉中加水,然后匀浆,热处理10~30 min后,再次匀浆待用。
其中,贝肉与水的用量比优选为1g:(3~8) mL。
其中,优选使用沸水浴进行热处理。
其中,贝肉中加的水优选为超纯水。
具体地,步骤S1中:酶解后,对酶解液进行分离纯化。
其中,所述的分离纯化包括使用超滤和纳滤对酶解液进行分级,获得一定分子量范围的肽,以及对分级后的组分进行凝胶分离。使用纳滤可脱除盐和游离氨基酸,使用超滤脱除大分子物质。凝胶分离中,优选使用AKTA蛋白纯化系统Superdex peptide 10/300 GL凝胶柱进行分离,凝胶分离后,对收集到的峰进行感官评价,并筛选。
其中,步骤S1中:分离纯化后,将产物冷冻干燥后备用。
进一步,步骤S2中:使用LC-MS/MS进行多肽序列分析,通过与海湾扇贝蛋白数据库进行比对分析,得出全部肽的序列。
具体地,步骤S2中,优选对步骤S1获得的产物先脱盐,再进行多肽序列分析。
其中,优选使用C18 StageTip色谱柱进行脱盐。
进一步,步骤S3中:优选使用Discovery Studio 2019软件筛选可与鲜味受体T1R1/T1R3有效结合的海湾扇贝肽。
本发明的目的之三是提供上述扇贝鲜味肽的应用。
具体地,上述扇贝鲜味肽可应用于调味品的制备和食品风味的改善。
本发明的有益效果如下:
本发明通过两步酶解从海湾扇贝肉中获得了一种鲜味肽,与鲜味受体T1R1/T1R3对接能量分别为-81.3746 kcal/mol、-100.45 kcal/mol、-90.3756 kcal/mol 和-102.943kcal/mol。本发明的鲜味肽有较低的鲜味阈值以及较强的增鲜提鲜作用,四种合成肽的鲜味阈值分别为0.30mg/mL、0.45mg/mL、0.25mg/mL、0.50mg/mL,增鲜阈值分别为0.15mg/mL、0.20 mg/mL、0.15 mg/mL、0.40 mg/mL。本发明的鲜味肽可用于调味品制作,改善食品风味,具有广阔的市场前景。
附图说明
图1为氨基酸序列如SEQ.ID.NO.1所示的鲜味肽与鲜味受体T1R1/T1R3的分子对接结果图;
图2为氨基酸序列如SEQ.ID.NO.2所示的鲜味肽与鲜味受体T1R1/T1R3的分子对接结果图;
图3为氨基酸序列如SEQ.ID.NO.3所示的鲜味肽与鲜味受体T1R1/T1R3的分子对接结果图;
图4为氨基酸序列如SEQ.ID.NO.4所示的鲜味肽与鲜味受体T1R1/T1R3的分子对接结果图;
图5为实施例中步骤S1中经过超滤、纳滤分级后获得的肽的凝胶色谱图;
图6为实施例中步骤S1凝胶色谱分离出的F1、F2、F3和F4四个组分的感官滋味轮廓图;
图7为测试1中四种鲜味肽分子的感官滋味轮廓图。
具体实施方式
以下结合实例对本发明的原理和特征进行描述,所举实例只用于解释本发明,并非用于限定本发明的范围。下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径获取。
实施例
制备扇贝鲜味肽,步骤如下:
S1. 制备海湾扇贝肽:
(1)原料预处理:称取海湾扇贝肉1 kg,加入5 L超纯水并匀浆,沸水浴加热15min,再次匀浆,得匀浆液待用。
(2)酶解:向步骤(1)获得的匀浆液中加入以贝肉计2000 U/g的AP-200碱性蛋白酶,调节pH 9,35 ℃酶解6 h,然后100℃煮沸灭酶;冷却后,加入以贝肉计600U/g的FF106风味蛋白酶,调节pH7,55 ℃酶解5 h,然后100℃煮沸灭酶;之后8000 r/min离心15min,取上清。
(3)纯化:
a. 超滤、纳滤分级:先采用200 Da纳滤膜除盐和游离氨基酸,之后选择3000 Da卷式膜进行超滤,将得到的200~3000 Da组分,冷冻干燥,-20 ℃保存备用。
b. 使用AKTA蛋白纯化系统Superdex peptide 10/300 GL凝胶柱进行分离;选择流动相为超纯水,肽样品浓度100 mg/mL,一次取0.5 mL 进样,设置洗脱流速为0.5 mL/min,设置自动收集时间为2 min每次;分别将每个分离峰的分离组分收集,分别标记为F1、F2、F3和F4(凝胶色谱图如图5所示),并进行感官评定,筛选出最佳组分为F1。感官评定结果见图6。
感官评定方法如下:参考ISO 8589-2007标准进行感官评价。为了对样品的5种基本味道(酸、甜、苦、咸、鲜)进行准确科学的感官评价,经过筛选,剔除不能识别五种基本味觉种类和强度的成员,选取 10 名人员组成感官评定小组(5名男性和5名女性,年龄20~30岁),感官实验在标准感官实验室进行。为了使感官评定小组成员获得统一的感官评价味感标准,小组成员接受2周内进行的3次时长2 h的培训。培训使用参比溶液酸/柠檬酸、甜/蔗糖、苦/硫酸奎宁、咸/氯化钠和鲜/味精分别以0.8 mg/mL、10 mg/mL、0.020 mg/mL、3.5 mg/mL、3.5 mg/mL为5分,以此为标准进行训练,使小组成员达成评价共识,感官评分分数即为味感强度值。
S2. 对海湾扇贝肽进行序列鉴定:
将步骤S1得到的F1组分肽粉使用C18 StageTip色谱柱脱盐,并使用LC-MS/MS进行多肽序列分析,通过与海湾扇贝蛋白数据库进行比对分析,得出全部肽序列。
S3. 鲜味肽与鲜味受体T1R1/T1R3的分子对接:
利用Discovery Studio 2019构建多肽结构,对所有多肽添加CHARMm力场,采用Smart Minimizer算法对其进行能量最小化处理,最大步数设定为2000步,RMS梯度值设为0.01;利用swissmodel构建蛋白结构。
使用Discovery Studio 2019 进行分子对接前的准备工作。对蛋白结构进行处理,质子化和加氢原子,最后利用Discovery Studio软件寻找结合位点;然后将准备好的配体小分子,利用Discovery Studio 软件的CDOCKER半柔性对接方式进行分子对接,其他参数设为默认,仅保留一个 C E最低的构象, 得到四个多肽序列的对接能量,四个多肽氨基酸序列依次如SEQ.ID.NO.1~4所示,鲜味肽的氨基酸序列与相应的对接能量如表1所示。
表1 鲜味肽氨基酸序列及对接能量
氨基酸序列如SEQ.ID.NO.1所示的鲜味肽与鲜味受体T1R1/T1R3的分子对接结果见图1,氨基酸序列如SEQ.ID.NO.2所示的鲜味肽与鲜味受体T1R1/T1R3的分子对接结果见图2,氨基酸序列如SEQ.ID.NO.3所示的鲜味肽与鲜味受体T1R1/T1R3的分子对接结果见图3,氨基酸序列如SEQ.ID.NO.4所示的鲜味肽与鲜味受体T1R1/T1R3的分子对接结果见图4。
通过四种鲜味肽和鲜味受体的结合模式和结合位点可知,鲜味肽与T1R3亚基的结合最主要是通过与受体氨基酸残基之间产生的氢键作用、静电相互作用。这四种鲜味肽与T1R1/T1R3亚基的结合位点主要为GLU-148、ASP-216、ASP-219、ASP-218。其中,氨基酸序列如SEQ.ID.NO.1所示的鲜味肽与T1R1/T1R3亚基的结合位点为GLU-148、ASP-216、ASP-219、LYS-155、SER-109、ASN-150、GLU-52、GLN-278;氨基酸序列如SEQ.ID.NO.2所示的鲜味肽与T1R1/T1R3亚基的结合位点为GLU-148、ASP-216、LYS-155、ASP-219、ASN-150、ASP-218、GLN-278、GLN-52;氨基酸序列如SEQ.ID.NO.3所示的鲜味肽与T1R1/T1R3亚基的结合位点为ASN-150、SER-107;氨基酸序列如SEQ.ID.NO.4所示的鲜味肽与T1R1/T1R3亚基的结合位点为GLN-221、GLU-217、GLU-148、ASP-216、GLN-222、SER-217、ASP-218。
测试1
对实施例制备获得的四种鲜味肽进行固相合成,并进行感官鉴定,参考ISO 8589-2007标准进行感官评价。为了对样品的5种基本味道(酸、甜、苦、咸、鲜)进行准确科学的感官评价,经过筛选,剔除不能识别五种基本味觉种类和强度的成员,选取10名人员组成感官评定小组(5名男性和5名女性,年龄20~30岁),感官实验在标准感官实验室进行。为了使感官评定小组成员获得统一的感官评价味感标准,小组成员接受2周内进行的3次时长2 h的培训。培训使用参比溶液酸/柠檬酸、甜/蔗糖、苦/硫酸奎宁、咸/氯化钠和鲜/味精分别以0.8 mg/mL、10 mg/mL、0.020 mg/mL、3.5 mg/mL、3.5 mg/mL为5分,以此为标准进行训练,使小组成员达成评价共识,感官评分分数即为味感强度值。
感官评定结果显示,四种肽均有不同强度的鲜味和甜味,其中SEQ.ID.NO.3(RPPVVR)的呈味特征在四种肽之中较为显著,可能是其中含有的甜味氨基酸缬氨酸引起,SEQ.ID.NO.2(RGFGGAR)在鲜味的基础上有一定程度的苦味,可能是由于苯丙氨酸引起。测试获得的感官滋味轮廓图见图7。
测试2
对实施例制备获得的四种肽的合成产物进行鲜味阈值和增鲜阈值测定,将起始质量浓度为1 mg/mL的鲜味肽溶液逐级稀释,采用三角试验法,直到评价人员无法品尝出溶液中的鲜味,鲜味肽的鲜味阈值即为上一个鲜味肽溶液的质量浓度值;在0.5 mg/mL味精溶液中添加鲜味肽,等比例提高溶液中合成肽的质量浓度,直至可以明显品尝到溶液中的鲜味增加,合成肽的增鲜阈值即为此时溶液的质量浓度值。测试结果见表2。
表2 鲜味肽的鲜味阈值和增鲜阈值
测试结果表明,本发明的鲜味肽有较低的鲜味阈值以及较强的增鲜提鲜作用。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (3)
1.一种扇贝鲜味肽,其特征在于,包括氨基酸序列如SEQ.ID.NO.1~4所示的肽中的至少一种。
2.一种如权利要求1所述的扇贝鲜味肽的制备方法,其特征在于,包括如下步骤:
S1. 获取扇贝肽:通过对海湾扇贝的贝肉进行酶解获得扇贝肽,酶解的工作条件如下:向待处理原料中加入碱性蛋白酶,调节pH 8~10,30~40 ℃酶解5~7 h,灭酶;冷却后,加入风味蛋白酶,调节pH 6~7,50~60 ℃酶解5~8 h,灭酶;
酶解后,对酶解液进行分离纯化;所述的分离纯化包括使用超滤和纳滤对酶解液进行分级,以及对分级后的组分进行凝胶分离;
S2. 对扇贝肽进行序列鉴定:使用LC-MS/MS进行多肽序列分析,通过与海湾扇贝蛋白数据库进行比对分析,得出全部肽的序列;
S3. 筛选与鲜味受体T1R1/T1R3结合的扇贝肽。
3.一种如权利要求1所述的扇贝鲜味肽在调味品制备或食品风味改善中的应用。
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